ABSTRACT
E2F4 is an important basal transcription factor with the potential to promote tumor growth. Its upregulation in various types of cancer has been linked to numerous genetic factors; however, the nature of the involvement of epigenetic mechanisms, including DNA methylation, remains elusive. In the present study, E2F4 expression profiles were determined in 100 paired breast tumor and control samples, through RT-qPCR using the SYBR® green method. Furthermore, the E2F4 promoter methylation status in each of these samples was assessed using methylation specific PCR, in order to evaluate its impact on gene expression. A two-fold increase in E2F4 gene expression was observed in the breast tumors compared with in their respective controls (P=0.022); of these tumors, ~72% were under-methylated. The change in methylation status was also significantly higher (P<0.001) in the tumor samples. Methylation status was negatively correlated (r=-30) with E2F4 expression profiles, indicating that a decrease in methylation may promote higher expression of E2F4. The two study cohorts (>45 and ≤45 years) had comparable methylation profiles, though they had significantly decreased methylation status compared with controls. Various histo-pathological types also have different methylation profiles, indicating the presence of a tissue specific methylation signature. The results of the present study demonstrated that E2F4 methylation status can have a notable influence on its expression, and that it may have prognostic value in breast carcinogenesis.
ABSTRACT
Dynamic positioning of nucleosomes is pivotal in determining level of genes expression especially on or around transcription start site (TSS) of a gene. Purpose of the current study was to determine nucleosome position around TSS of Rbl2/p130. We investigated Rbl2/p130 expression in connection to nucleosome positions around its TSS among breast tumors and their adjacent normal control tissues (ANCT) using micrococcal nuclease (MNAse) digestion assay and ChIP-PCR analysis. Three fold reduced Rbl2/p130 expression in these tumor tissues were noticed compared to their control tissues. DNA obtained from MNAse digested chromatin was used as PCR template. Region between - 137 to + 140 around TSS was scanned using 3 primer pairs (P1 = - 137 to + 69; P2 = - 90 to + 69; P3 = - 33 to + 140). ~ 66% breast tumors and ~ 26% ANCT samples were positive for P1. The difference was found statistically significant (p = 0.000) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~ 9 times more probable in tumor samples. ~ 73% of the tumor and 60% ANCT were positive for P2, which although is significant (p = 0.035) with OD = 3.250, but less preferable than P1. However, P3 was not found to be a preferred area for nucleosome occupancy (p = 0.670; OD = 1.2). Negative correlations for nucleosome positions were observed especially for P1. Our results indicate that nucleosome are present slightly downstream of TSS in routine, while in case of breast carcinogenesis nucleosomes slides 55 bases upstream of the TSS, aligning + 1 position at the center of nucleosome, hence hindering access to the transcriptional machinery.
